We have shown that RGS proteins modulate signaling through a variety of G-protein coupled receptors including chemokine receptors. Chemokine receptors signal predominantly by triggering Galphai nucleotide exchange. Humans and mice have three Galphai isoforms although Galphai2 (encoded by Gnai2) and Galphai3 (encoded by Gnai3) predominate in lymphoid cells. We have found that Gnai2-/- T and B cells have severe defects in chemokine-receptor signaling. In vivo, the Gnai2-/- B cells fail to properly access lymph node follicles and the Gnai2-/- T cells fail to properly enter the T cell zone. We have studied the immune system with a focus on immune cell trafficking in various RGS deficient mice. We have supplemented these studies with in vitro studies using mRNA knock-downs as well as over expression studies using RGS proteins fused to a fluorescent marker. The recognition of the importance of both G-protein signaling and RGS proteins in the regulation of lymphocyte responses to chemokines has led us to study other hematopoietic cell types. Rgs13 is highly expressed in germinal center B cells. Germinal centers are the site of affinity maturation of the antibody response. B cells with high affinity B cell receptors are selected to expand in germinal centers. To better understand the role of Rgs13 in germinal center B cells we have developed mice that lack Rgs13 as well as mice that express the Cre recombinase under control of the Rgs13 regulatory sequences. By crossing the Rgs13-Cre mice with the R26R-confetti mice we can monitor those cells that have expressed Rgs13. Preliminary analysis of these mice revealed expression of multiple colored GC B cells. Current studies are examining the fate of these cells during B cell differentiation to plasma and memory B cells. RGS proteins work in conjunction with the beta-arrestin proteins to regulate chemokine and sphingosine-1 phosphate receptor signaling in lymphocytes. To assess the importance of beta-arrestin1 and beta-arrestin2 in lymphocyte function we have acquired mice that lack either beta-arrestin1 or beta-arrestin2. The analysis of these mice suggests significant problems in the sphingosine-1 phosphate receptor 1 (S1PR1) signaling pathway. Intriguingly the humoral immune response is significantly impaired in the beta-arrestin2 mice. To explore the interaction between the S1PR1 receptor and the beta-arrestin proteins we have established a BRET assay, which will be used to assess the recruitment of these proteins to S1PR1 and to the chemokine receptor CXCR5, one of the major B cell chemoattractant receptors. Besides Galphai proteins B lymphocytes also strongly express Galphas. To assess the importance of Galphas signaling in B lymphocytes we have acquired mice that have a floxed Galphas allele. These mice have been backcrossed on to a C57Bl/6 background and then crossed to mice with a knock-in of the Cre recombinase under the control of the mb1 regulatory sequences. This allows for a B lymphocyte specific deletion of Galphas. These mice are now available for analysis. Preliminary results suggest problems in B cell development and differentiation in the spleen. Further functional studies are in progress. We have examined the role of Galphai proteins in the follicular (FO) versus marginal zone (MZ) B cell fate decision in the spleen. Mice lacking B cell expression of Galphai2 and Galphai3 exhibit a striking decrease in MZ B cell development. We found that direct contact with Notch ligand expressing stromal cells (OP9-DL1) could not rescue this developmental defect. Briefly treating wild type B cell progenitors with pertussis toxin, which irreversibly blocks subsequent Galphai nucleotide exchange, inhibited the appearance of MZ B cells in OP9-DL1 cultures. Revealing faulty Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) dependent Notch2 processing, Galphai deficient transitional B cells constitutively had low membrane ADAM10 expression and reduced levels of Notch2 target genes despite normal Notch2 levels. Immunoblotting B cell lysates from Galphai deficient B cells documented a defect in the processing of ADAM10 to its mature form. Suggesting that Galphai signaling promotes ADAM10 membrane expression, stimulating wild type transitional B cells with CXCL12 raised it. These results showed that Galphai signaling supports ADAM10 maturation and activity in developing splenic B cells, and ultimately Notch2 signaling to help drive MZ B cell development.